A Flavobacterium with siderophore production, growth promotion and alleviation of plant salt-alkali stress and its application

Abstract

The present invention discloses a Flavobacterium strain with the ability to produce siderophores, promote growth and alleviate saline-alkali stress in plants, and its application. The present invention provides Flavobacterium conyzicola ( Flavobacterium erigeronis ), with the strain number 3-218, and its registration number in the China General Microbiological Culture Collection Center is CGMCC No. 31149. Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3-218 can be used as a microbial organic fertilizer to improve soil fertility, alleviate saline-alkali stress in crops, and enhance the saline-alkali adaptability of crops.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a Flavobacterium strain with the functions of producing siderophores, promoting plant growth and alleviating plant saline-alkali stress, and its application. Background Art

[0002] The rhizosphere microbiome plays a key role in plant growth and health, improving nutrient utilization efficiency, and protecting plants from biotic and abiotic stresses. Plant growth-promoting rhizobacteria (PGPR) have gradually been used as biological bacterial agents and play an increasingly important role in agricultural production. In recent years, studies have shown that PGPR can not only promote plant growth, prevent diseases, and increase crop yields, but also improve the resistance of plants to various abiotic stresses such as drought, salt, and heavy metals, and enhance the adaptability of plants to various environmental stresses.

[0003] Numerous PGPR groups have been discovered at home and abroad, which have functions such as secreting plant hormones, dissolving phosphorus, dissolving iron, and decomposing potassium. The genus Flavobacterium ( Flavobacterium ) is a type of Gram-negative bacteria widely present in the environment, and has been reported in promoting plant growth, stress resistance (drought, cold, etc.), and sewage treatment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new strain of the genus Flavobacterium ( Flavobacterium ) with the functions of producing siderophores, promoting plant growth and alleviating plant saline-alkali stress.

[0005] In the first aspect, the present invention claims protection for Flavobacterium conyzicola ( Flavobacterium erigeronis ).

[0006] The strain number of Flavobacterium conyzicola ( Flavobacterium erigeronis ) claimed by the present invention is 3-218, and its registration number in the China General Microbiological Culture Collection Center is CGMCC No. 31149.

[0007] Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3-218 is a Gram-negative bacterium. After growing on R 2 A solid medium for 3 days, the colonies are yellow, round, convex, with mucus, easy to pick, and the colony diameter is 1-2 mm. This strain has excellent plant growth promotion ability and can significantly alleviate the stress of saline-alkali on plants.

[0008] In the second aspect, the present invention claims protection for the culture of Flavobacterium conyzicola ( Flavobacterium erigeronis ) described in the first aspect above.

[0009] The culture claimed by the present invention is the Flavobacterium conyzicola ( Flavobacterium erigeronis Substances obtained by culturing in a bacterial medium (all substances in the culture vessel, i.e., fermentation products, such as containing Flavobacterium conyzicola ( Flavobacterium erigeronis 3-218) and substances secreted into the liquid medium, i.e., fermentation broth, or such as containing Flavobacterium conyzicola ( Flavobacterium erigeronis 3-218) and substances secreted into the solid medium, i.e., solid fermented products).

[0010] Among the above-mentioned cultures, the substances include Flavobacterium conyzicola ( Flavobacterium erigeronis )(the cells themselves) and its metabolites described in the first aspect above.

[0011] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and generate substances and energy for maintaining life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monomers like monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, etc., and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process by which microorganisms synthesize some substances with no clear function for the life activities of microorganisms using primary metabolites as precursors during a certain growth period. The products of secondary metabolism are secondary metabolites, mostly compounds with relatively complex molecular structures. According to their functions, they can be classified into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0012] Among the above-mentioned cultures, the bacterial medium can be a solid medium or a liquid medium.

[0013] The term "culture" refers to the general term for liquid or solid media with a microbial population grown after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms or can contain a certain amount of medium, metabolites, or other components produced during the culture process. The term "culture" also includes subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0014] In the third aspect, the present invention claims to protect a bacterial agent.

[0015] The bacterial agent claimed to be protected by the present invention contains Flavobacterium conyzicola ( Flavobacterium erigeronis ) described in the first aspect above, the metabolites of the Flavobacterium conyzicola ( Flavobacterium erigeronis ) and / or the culture described in the second aspect above.

[0016] Among them, the bacterial agent can be a bacterial agent for alleviating plant salt-alkali stress.

[0017] The microbial agent also has all or part of the following functions: secreting siderophores, enhancing the antioxidant enzyme activity of plants, increasing the proline content of plants, promoting plant growth (such as increasing plant stem diameter, enhancing plant fresh weight and / or dry weight, increasing plant height), and promoting plant seed germination (such as enhancing plant germination potential and / or germination rate).

[0018] In the above microbial agent, the active ingredient of the microbial agent can be the Flavobacterium conyzicola described in the first aspect above ( Flavobacterium erigeronis ), the metabolite of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), and / or the culture of the Flavobacterium conyzicola ( Flavobacterium erigeronis ). The active ingredient of the microbial agent can also contain other biological components or / and non-biological components. Those skilled in the art can determine other active ingredients of the microbial agent according to the desired effects.

[0019] In the above microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier can be a carrier commonly used in the pesticide field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material, or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, bean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0020] In the above microbial agent, the dosage form of the microbial agent can be various dosage forms, such as a liquid agent, an emulsion, a suspension, a powder, a granule, a wettable powder, or a water dispersible granule.

[0021] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the microbial agent.

[0022] In the microbial agent, the Flavobacterium conyzicola ( Flavobacterium erigeronis ), strain 3-218, or / and the metabolite of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), strain 3-218, can exist in the form of cultured live cells, the fermentation broth of live cells, the filtrate of cell cultures, or a mixture of cells and filtrates.

[0023] In the fourth aspect, the present invention claims any one of the following applications of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), or the metabolite of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), described in the first aspect above, or the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0024] (A1) Secreting siderophores;

[0025] (A2) Preparing a product for secreting siderophore;

[0026] (A3) Increasing the activity of plant antioxidant enzymes;

[0027] (A4) Preparing a product for increasing the activity of plant antioxidant enzymes;

[0028] (A5) Increasing the proline content of plants;

[0029] (A6) Preparing a product for increasing the proline content of plants.

[0030] In a fifth aspect, the present invention claims any one of the following applications of Flavobacterium conyzicola described in the first aspect above ( Flavobacterium erigeronis ), or metabolites of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), or the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0031] (B1) Promoting plant growth;

[0032] (B2) Preparing a product for promoting plant growth;

[0033] (B3) Promoting the germination of plant seeds;

[0034] (B4) Preparing a product for promoting the germination of plant seeds.

[0035] In a sixth aspect, the present invention claims any one of the following applications of Flavobacterium conyzicola described in the first aspect above ( Flavobacterium erigeronis ), or metabolites of the Flavobacterium conyzicola ( Flavobacterium erigeronis ), or the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0036] (C1) Increasing the germination potential and / or germination rate of plants;

[0037] (C2) Preparing a product for increasing the germination potential and / or germination rate of plants;

[0038] (C3) Increasing the stem diameter of plants;

[0039] (C4) Preparing a product for increasing the stem diameter of plants;

[0040] (C5) Increasing the fresh weight and / or dry weight of plants;

[0041] (C6) Preparing a product for increasing the fresh weight and / or dry weight of plants;

[0042] (C7) Increasing the plant height;

[0043] (C8) Preparing a product for increasing the plant height.

[0044] In the fourth aspect described above, the improvement of the antioxidant enzyme activity of plants can be the improvement of the antioxidant enzyme activity of plants under alkaline stress conditions. The increase in the proline content of plants is the increase in the proline content of plants under alkaline stress conditions.

[0045] In the fifth aspect described above, the promotion of plant growth can be the promotion of plant growth under alkaline stress conditions; the promotion of plant seed germination can be the promotion of plant seed germination under alkaline stress conditions.

[0046] In the sixth aspect described above, the improvement of the germination potential and / or germination rate of plants can be the improvement of the germination potential and / or germination rate of plants under alkaline stress conditions; the increase in the stem diameter of plants can be the increase in the stem diameter of plants under alkaline stress conditions; the increase in the fresh weight and / or dry weight of plants can be the increase in the fresh weight and / or dry weight of plants under alkaline stress conditions; the increase in the plant height of plants can be the increase in the plant height of plants under alkaline stress conditions.

[0047] In the fourth aspect described above, the antioxidant enzyme can be catalase (CAT) or peroxidase (POD).

[0048] In the seventh aspect, the present invention claims any one of the following applications of Flavobacterium conyzicola ( Flavobacterium erigeronis ), or the metabolite of Flavobacterium conyzicola ( Flavobacterium erigeronis ), or the culture described in the second aspect above, or the microbial inoculum described in the third aspect above:

[0049] (D1) Alleviating saline-alkali stress of plants;

[0050] (D2) Preparing a product for alleviating saline-alkali stress of plants;

[0051] (D3) Improving soil fertility;

[0052] (D4) Preparing a product for improving soil fertility.

[0053] In each of the above relevant aspects, the product can be a microecological preparation or a biological fertilizer.

[0054] The present invention also provides a method for preparing the microbial inoculum described in the third aspect above.

[0055] The method for preparing the microbial inoculum provided by the present invention includes using Flavobacterium conyzicola ( Flavobacterium erigeronis ), or / and the metabolite of Flavobacterium conyzicola ( Flavobacterium erigeronis ), or / and the above culture as components of the microbial inoculum to obtain the microbial inoculum.

[0056] In this article, the metabolite of Flavobacterium conyzicola ( Flavobacterium erigeronis ), or / and the above culture as components of the microbial inoculum to obtain the microbial inoculum. Flavobacterium erigeronis ), obtained from the fermentation broth of 3 - 218. The Flavobacterium canadense ( Flavobacterium erigeronis ), the metabolite of 3 - 218 can be the sterile metabolite of Flavobacterium canadense ( Flavobacterium erigeronis ), or the bacteria - containing metabolite of Flavobacterium canadense ( Flavobacterium erigeronis ). The sterile metabolite (sterile fermentation filtrate) of Flavobacterium canadense ( Flavobacterium erigeronis ), can be prepared specifically according to the following method: Cultivate Flavobacterium canadense ( Flavobacterium erigeronis ) in a liquid medium, and filter out Flavobacterium canadense ( Flavobacterium erigeronis ) from the liquid culture (fermentation broth), then the sterile metabolite of Flavobacterium canadense ( Flavobacterium erigeronis ) is obtained. The bacteria - containing metabolite of Flavobacterium canadense ( Flavobacterium erigeronis ) can be prepared specifically according to the following method: Cultivate Flavobacterium canadense ( Flavobacterium erigeronis ) in a liquid fermentation medium, and collect the fermentation broth - containing Flavobacterium canadense ( Flavobacterium erigeronis ) and the substances secreted into the liquid medium, and this fermentation broth is the bacteria - containing metabolite of Flavobacterium canadense ( Flavobacterium erigeronis ).

[0057] The present invention also provides any of the following methods:

[0058] Method I: A method for alleviating plant salt - alkali stress, comprising the following steps: Treat the plant to be treated or its growth substrate with the Flavobacterium canadense ( Flavobacterium erigeronis ) or the metabolite of the Flavobacterium canadense ( Flavobacterium erigeronis ) described in the first aspect above, or the culture described in the second aspect above, or the microbial agent described in the third aspect above, so as to alleviate plant salt - alkali stress.

[0059] Method II: A method for promoting plant growth under alkali stress conditions, comprising the following steps: Treat the plant to be treated or its growth substrate with the Flavobacterium canadense ( Flavobacterium erigeronis ) or the metabolite of the Flavobacterium canadense ( Flavobacterium erigeronis ) described in the first aspect above, or the culture described in the second aspect above, or the microbial agent described in the third aspect above, so as to promote plant growth under alkali stress conditions.

[0060] Method III: A method for promoting plant seed germination under alkali stress conditions, comprising the following steps: Treat the plant to be treated or its growth substrate with the Flavobacterium canadense ( Flavobacterium erigeronis ) or the Flavobacterium canadense (Flavobacterium erigeronis The metabolites described in (2)), the cultures described in the second aspect above, or the bacterial agents described in the third aspect above are used to treat the plant seeds to be treated (such as soaking the seeds or mixing them with the seeds), so as to promote the germination of plant seeds under alkaline stress conditions.

[0061] In each of the above related aspects, in one embodiment of the present invention, the saline-alkali stress is simulated as follows: NaCl, Na with a molar ratio of 1:1:1 2 SO 4 and NaHCO 3 , Na + A mixed solution with a final concentration of 168 mmol / L.

[0062] In each of the above related aspects, the plant may be any one of the following:

[0063] (E1) Angiosperms;

[0064] (E2) Monocotyledonous plants;

[0065] (E3) Plants of the order Poales;

[0066] (E4) Gramineous plants;

[0067] (E5) Plants of the subfamily Panicoideae;

[0068] (E6) Plants of the tribe Maydeae;

[0069] (E7) Zea mays (commonly known as corn).

[0070] Through the test of siderophore secretion of the present invention, Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 has the ability to secrete siderophores. The pot experiment shows that compared with the un-inoculated negative control group, after inoculating with Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218, it can increase the stem diameter, plant height, fresh weight and dry weight of Zea mays, and significantly improve the activities of antioxidant enzymes and the content of proline in corn. Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 can be used as a microbial organic fertilizer to improve soil fertility, relieve saline-alkali stress of crops, and enhance the saline-alkali adaptability of crops.

[0071] Depositing Instructions

[0072] Taxonomic name: Flavobacterium canadense ( Flavobacterium erigeronis );

[0073] Reference biological material: 3-218;

[0074] Depository institution: China General Microbiological Culture Collection Center;

[0075] Abbreviation of the depositary institution: CGMCC;

[0076] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0077] Date of deposit: July 3, 2024;

[0078] Accession number registered by the depositary center: CGMCC No. 31149. Description of the drawings

[0079] Figure 1 Colony morphology of Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 after 3 days of cultivation on RA plates. 2

[0080] Figure 2 Phylogenetic tree of Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 and related type strains constructed by the neighbor-joining method based on 16S rRNA gene sequences. Note: The GenBank accession numbers of the 16S rRNA gene sequences of the strains are shown in parentheses; the reference strains in the figure are all type strains of their respective species. Sphingobacterium spiritivorum ATCC 33861 T (ACHA01000008) was used as the outgroup.

[0081] Figure 3 Protein sequence phylogenetic tree of Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 and closely related species within the genus constructed by the maximum likelihood method based on 836 orthologous genes. Flavobacterium

[0082] Figure 4 Results of the detection of the ability of Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 to secrete siderophores. CK is the control without inoculating the bacterial solution.

[0083] Figure 5 Growth status of maize seedlings inoculated with strain 3-218 and non-inoculated strains under saline-alkali stress at 30 days.

[0084] Figure 6 Fresh weight, dry weight, plant height and root length of maize seedlings under 30-day saline-alkali stress. CK is the control without inoculating the bacterial solution. * in the figure indicates significant difference ( P < 0.05).

[0085] Figure 7 Stem diameter and chlorophyll content of maize seedlings under 30-day saline-alkali stress. CK is the control without inoculating the bacterial solution. * in the figure indicates significant difference ( P < 0.05).​​ Detailed implementation manners

[0086] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0087] In the following experimental methods of the following embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0088] R 2 Liquid A medium: 0.25 g of tryptone, 0.5 g of acid-hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5 g of glucose, made up to 1000 mL with water, pH value 7.2 ± 0.2, sterilized at 121 °C for 15 min.

[0089] R 2 Solid A medium: R 2 20.0 g of agar was added to Liquid A medium and sterilized at 121 °C for 15 min.

[0090] TSB liquid medium: 15.0 g of tryptone, 5.0 g of soy peptone, 5.0 g of sodium chloride, 1000 mL of distilled water, adjusted to pH 7.3 ± 0.2, sterilized at 121 °C for 15 min.

[0091] TSA solid medium: 20.0 g of agar was added to TSB liquid medium and sterilized at 121 °C for 15 min.

[0092] Starch medium: NA medium, 0.2% soluble starch (% means g / 100 mL), pH 7.4, sterilized at 121 °C for 20 min.

[0093] Casein medium: Solution a: 5 g of skim milk powder, 50 mL of distilled water; Solution b: 50 mL of NB, 1.5 g of agar. Solution a and solution b were sterilized at 121 °C for 15 min respectively. After cooling to about 60 °C, they were mixed evenly and dispensed into plates.

[0094] CAS qualitative medium:

[0095] Solution ①: 0.012 g of CAS was dissolved in 10 mL of deionized water and mixed with 2 mL of 5 mM ferric chloride;

[0096] Solution ②: 0.015 g of cetyltrimethylammonium bromide is dissolved in 8 mL of deionized water;

[0097] Dye solution ③: Slowly pour Solution ① into Solution ② to obtain Dye solution ③, and sterilize it at 115 °C for 20 min;

[0098] Culture medium ④: Add 6.04 g of piperazine diethanol sulfonic acid and 10 mL of 0.1 M phosphate solution to a triangular flask containing 150 mL of distilled water and mix well. Adjust the pH to 6.8 with 50% NaOH, add 4.0 g of agar powder, and sterilize it at 115 °C for 20 min;

[0099] Phosphate solution: 2.427 g of disodium hydrogen phosphate, 0.5905 g of sodium dihydrogen phosphate, 0.075 g of potassium dihydrogen phosphate, 0.125 g of sodium chloride, 0.25 g of ammonium chloride, 100 mL of deionized water, mix evenly, and dilute 10 times before use;

[0100] Nutrient solution: 0.2 mL of 1 mM calcium chloride solution, 4 mL of 1 mM magnesium sulfate tetrahydrate solution, 6 mL of 10% (w / V) casein amino acid solution, respectively sterilize at 115 °C for 20 min (10 mL can be prepared when in use, and store it in a 4 °C refrigerator in the dark for future use after use);

[0101] CAS qualitative medium plate: When the dye solution, Culture medium ④, and nutrient solution are cooled to about 65 °C, add the nutrient solution to Culture medium ④, and then slowly add Dye solution ③. Mix well and pour the plate.

[0102] CAS detection solution: 10 mM cetyltrimethylammonium bromide, 1.5 mL of 1 mM ferric chloride, 7.5 mL of 2 mM CAS, 4.307 g of anhydrous bis(dimethylamine), adjust the pH with 12 mM HCl to completely dissolve the anhydrous bis(dimethylamine), and 100 mL of deionized water.

[0103] Example 1. Isolation and identification of Flavobacterium conyzicola Flavobacterium erigeronis 3-218

[0104] I. Isolation of Flavobacterium conyzicola Flavobacterium erigeronis 3-218

[0105] Collect the rhizosphere soil sample of Conyza canadensis along the Lhasa Duilong River (29°48′8″N, 93°54′32″E), use a 4 °C ice box, and bring it back to the laboratory for storage in a 4 °C refrigerator.

[0106] Shake off the soil attached to the plant roots, and only retain the rhizosphere soil tightly adhered to the root surface. Immerse the Conyza canadensis roots with rhizosphere soil in a conical flask containing 100 mL of sterile water, and oscillate at 150 rpm at room temperature in a shaker for 30 min. Centrifuge the collected suspension at 3000 rpm for 10 min at 4°C. After discarding the supernatant, the remaining part is the rhizosphere soil. Weigh 1 g of rhizosphere soil and resuspend it in 10 mL of sterile water for serial dilution. Take 100 μL of the serial dilution solution and spread it on the R 2 A plate, and incubate it upside down at 30°C for 1 week. According to the physiological and morphological characteristics, use a bamboo stick to pick single colonies and inoculate them on the plate for purification. After determining them as pure bacteria, transfer them to a slant for short-term storage at 4°C and transfer them to a 20% glycerol tube for long-term storage at -80°C. Name one of the isolated and purified strains as 3-218.

[0107] II. Identification of Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3-218

[0108] 1. Strain morphological identification

[0109] Describe the single colony state of the strain 3-218 obtained by separation and purification in the above step 1, which is in the logarithmic growth phase and has a stable colony size. Mainly include the size, color, transparency, surface state of the colony, and edge state of the colony. According to the manufacturer's instructions, use the Gram staining kit of Solarbio Science & Technology Co., Ltd. (Solarbio) in Beijing to perform smear Gram staining on the strain 3-218, and observe the morphology of the bacteria using an optical microscope.

[0110] The colonies of strain 3-218 on the R 2 A plate are yellow, round, convex, with mucus, easy to pick, and the colony diameter is 1-2 mm ( Figure 1 ). The cells are Gram-negative, rod-shaped, and no endospores are formed.

[0111] 2. Molecular identification

[0112] Operate according to the instructions of the manual. Extract genomic DNA using the TIANamp Bacteria Genomic DNA Kit of TIANGEN Biochemical Technology (Beijing) Co., Ltd. (TIANGEN), and use the bacterial universal primers 27F (SEQ ID No.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID No.2: 5'-GGTTACCTTGTTACGACTT-3') to amplify the 16S rRNA gene. 50 μL PCR amplification system: 2×Taq PCR Mix 25 μL, 27F (10 μmol / L) 2 μL, 1492R (10 μmol / L) 2 μL, ddH 2O 19μL, 2μL DNA template. The PCR amplification program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 1min, 72℃ extension for 90s, 30 cycles; 72℃ final extension for 10min. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the amplified fragment was about 1300bp. After electrophoresis verification, the positive PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequence obtained by sequencing was uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment.

[0113] The sequencing length of the 16S rRNA gene of strain 3-218 was 1300 bp (SEQ ID No. 3). Comparison with the EzBioCloud database showed that strain 3-218 was a member of the genus Flavobacterium ( Flavobacterium ) members, and Flavobacterium chungangensis MAH-10 T (97.99%)、 Flavobacterium ustbae T13 T (97.84%)、 Flavobacterium ginsenosidimutans THG 01 T (97.49%) and Flavobacterium luteolum IMCC 34776 T (97.40%) sequences have a high similarity with Flavobacterium The sequence similarity of the model bacteria of other species of the genus was <98.8%. The closely related 16S rRNA gene sequences were retrieved from the EzBioCloud server and aligned using the MUSCLE program. The phylogenetic tree was constructed by the neighbor-joining method using the MEGA X software. The evolutionary distance of the NJ method was calculated using the Kimura two-parameter model, and the bootstrap value was 1000. The phylogenetic tree constructed by the neighbor-joining method is shown in Figure 2. Figure 2 As shown, strain 3-218 and strain Flavobacterium ustbae T13 T (MH593838) and Flavobacterium The strains of the genus were clustered together and formed a separate branch, indicating that strain 3-218 was Flavobacterium A potential new species of the genus.

[0114] 3. Genomic analysis

[0115] The genomic DNA was sent to Annoroad Gene Technology (Beijing) Co., Ltd., and the draft genome sequencing of strain 3-218 was carried out using the Illumina NovaSeq 6000 sequencing system. Assembly was performed using the SPAdes software, resulting in 126 contigs with a coverage of approximately 150×, and the N 50 length was 510,143 bp. The genome size was 5.54 Mb, and the G+C content was 34.60%. The average nucleotide identity (ANI) analysis of the whole genomes between strain 3-218 and Flavobacterium closely related strains of the genus was performed using the ANIm method in the pyANI software. The digital DNA-DNA hybridization (dDDH) value between strain 3-218 and the reference strain was calculated using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#).

[0116] Table 1. ANI and dDDH values of Flavobacterium canadense Flavobacterium erigeronis () 3-218 and Flavobacterium closely related species within the genus

[0117]

[0118] Compared with the type strains of other species of the genus with publicly available genomic sequences, the ANI value of strain 3-218 was 84-91%, lower than the critical values of 95-96% previously proposed for species delimitation; the dDDH values of strain 3-218 and its type strain were between 20.0-38.9%, far lower than the species delimitation threshold of 70%. See Table 1 for details. The results of both ANI and dDDH indicated that strain 3-218 was Flavobacterium a new species of the genus. Flavobacterium

[0119] At the same time, the Bacterial Pan Genome Analysis tool (BPGA) software was used to analyze strain 3-218 and Flavobacterium 32 type strains of the genus. A total of 1401 orthologous genes were concatenated, and a phylogenetic tree of protein sequences was constructed using the maximum likelihood method ( Figure 3 ), with a Bootstrap value of 1000. The results showed that strain 3-218 clustered together with strain Flavobacterium ustbae T13 T (GCA_003946915.1) and several strains of the genus, and formed a separate branch, indicating that strain 3-218 was Flavobacterium a potential new species of the genus. Flavobacterium

[0120] ​​4. Physiological and Chemical Classification and Identification

[0121] Add several drops of 5% H 2 0 2 onto a glass petri dish. Pick the strain 3-218 and react it with the above. If bubbles are generated, it proves that the strain can produce catalase. Spot inoculate the strain 3-218 on a filter paper soaked with 1% p-dimethylaminobenzene hydrochloride. Use Pseudomonas aeruginosa and Escherichia coli as positive and negative controls respectively. If a rosy red circle appears around the colony, it indicates that it can produce oxidase. Spot inoculate the strain 3-218 at 5 points on a starch medium, set 3 parallels, and culture at 25 °C for 2-5 d. After taking out the plate, add iodine solution dropwise around the colony and observe the color change around the colony. If there is a colorless transparent circle around the colony, it means that the bacterium produces amylase and diffuses into the matrix, and has hydrolyzed the starch in the medium into a substance that does not show color when reacting with iodine; if the area around the colony is blue, it means that the bacterium does not produce amylase. Spot inoculate the strain 3-218 at 5 points on a casein medium, set 3 parallels, and culture at 25 °C for 7 d. After taking out the plate, observe whether the casein around and under the colony is decomposed into a transparent circle. If it is transparent, it means that the strain has the ability to hydrolyze casein.

[0122] The results show that: when the strain 3-218 contacts with 5% H 2 0 2 , bubbles are generated, indicating that its catalase result is positive; when the strain 3-218 contacts with the filter paper soaked with 1% p-dimethylaminobenzene hydrochloride, a relatively light rosy red color appears on the bacteria, and its oxidase result is judged to be weakly positive; the strain produces transparent circles on both the casein medium and the starch medium with iodine solution added, indicating that the strain 3-218 has the ability to hydrolyze casein and starch.

[0123] Use the API 20NE, ZYM and 50 CH test strips (bioMérieux) of bioMérieux, France to determine the enzyme activity reactions and carbohydrate utilization of the strain 3-218 and related reference strains.

[0124] The results of the 20NE test show that: the glucosidation and esculin hydrolysis reactions of the strain 3-218 are positive, and it can assimilate glucose, arabinose, mannose, N N-acetyl-glucosamine and maltose. The nitrate reduction reaction, indole reaction, arginine hydrolysis reaction, urease hydrolysis reaction, gelatin hydrolysis reaction, p-nitro- β β-D-galactosidase hydrolysis reaction are negative, and it cannot assimilate mannitol, gluconate, capric acid, adipic acid, malic acid, citric acid, phenylacetic acid and tetramethyl-p-phenylenediamine.

[0125] In the ZYM enzyme activity identification test, alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, acid phosphatase, and N N-acetyl-glucosaminidase showed positive results; lipase (C8), lipase (C14), trypsin, chymotrypsin, naphthol-AS-BI-phosphohydrolase, α β-galactosidase, β α-galactosidase, β α-glucuronidase, α β-glucosidase, β α-glucosidase, α α-mannosidase, and α α-fucosidase showed negative results; cystine arylamidase showed a weakly positive result.

[0126] The 50 CH results showed that strain 3-218 could hydrolyze L-arabinose, D-xylose, galactose, glucose, fructose, mannose, N N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, starch, glycogen, and d-gentiobiose; it could not hydrolyze mannitol, erythritol, ribose, L-xylose, adonitol, β- methyl-d-xyloside, sorbose, dulcitol, inositol, mannitol, sorbitol, methyl- α α-d-mannopyranoside, methyl- α β-d-glucopyranoside, sucrose, trehalose, inulin, melezitose, raffinose, xylitol, d-turanose, d-lyxose, d-tagatose, d-fucose, L-fucose, d-arabitol, L-arabitol, gluconate, 2-keto-gluconate, and 5-keto-gluconate; it could weakly utilize d-arabinose, l-rhamnose, and d-melibiose. The physiological and biochemical characteristics differences between strain 3-218 and related type strains are shown in Table 2.

[0127] Table 2. Physiological and biochemical characteristics differences between strain 3-218 and related type strains

[0128]

[0129] Note: +, positive or utilizable; -, negative or non-utilizable; w, weakly positive.

[0130] Based on the homology alignment, construction of the phylogenetic tree, combined with the results of morphological, physiological and biochemical identification, and genomic analysis, it can be determined that strain 3-218 is a Flavobacterium new species of the Flavobacterium erigeronis genus, and the proposed taxonomic name is Flavobacterium erigeronis)3 - 218 was deposited in the China General Microbiological Culture Collection Center on July 3, 2024, with the registration number CGMCC No. 31149. Hereinafter referred to as Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218.

[0131] Example 2. Detection of the ability of Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 to secrete siderophores

[0132] Spot inoculate Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 onto the CAS qualitative medium and culture at 25 °C for 4 d. Observe whether there is an orange - yellow ring. If so, it preliminarily indicates that the strain has the ability to secrete siderophores.

[0133] Inoculate Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 into R 2 A liquid medium, and culture in the dark at 25 °C at 150 rpm for 2 d. Centrifuge at 12,000 rpm and take 1 mL of the supernatant. Mix it with an equal volume of CAS detection solution and let it stand in the dark at room temperature for 1 h. A is the absorbance value of the mixed solution at 630 nm, and Ar is the absorbance value of the mixed solution of the CAS detection solution and R 2 A liquid medium (1:1) at 630 nm. Zero with double - distilled water. Evaluate the ability of the strain to produce siderophores using the ratio of A to Ar. 0 - 0.6 indicates strong, 0.6 - 0.8 indicates medium, and 0.8 - 1 indicates weak (Yang Hongru, Yuan Bo, Zhao Xia, et al. Cultivable nitrogen - fixing bacterial groups in the rhizosphere of three desert shrubs and their nitrogen - fixing and siderophore - producing abilities [J]. Microbiology China, 2016, 43(11): 2366 - 2373. DOI: 10.13344 / j.microbiol.china.150967).

[0134] The results showed that after growing on the CAS qualitative medium for 4 d, Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 was able to produce an orange - yellow transparent ring, and the quantitative result of its siderophore - secreting ability was 0.62 ± 0.02 ( Figure 4 ).

[0135] Example 3. Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 alleviates plant salt - alkali stress

[0136] 1. Promote the germination of maize seeds under salt - alkali stress

[0137] Pick a single colony of Flavobacterium conyzicola ( Flavobacterium erigeronis )3 - 218 and inoculate it into a 200 mL R 2In a 500 mL conical flask of A liquid medium, culture at 25 °C for 48 h. Centrifuge at 8000 rpm for 10 min, collect the bacterial cells, and resuspend them with R 2 A liquid medium to form a bacterial suspension with an OD 600 = 1.0 (using the uninoculated R 2 A liquid medium as the control group for seed soaking). Select plump and large maize (Zhengdan 958) seeds and place them in the bacterial suspension, soak the seeds at 30 °C for 4 h.

[0138] Lay the soaked seeds flat on filter paper moistened with saline-alkali solution (composition: a mixed solution of NaCl, Na 2 SO 4 and NaHCO 3 with a molar ratio of 1:1:1, and the final concentration of Na + is 168 mmol / L). Place the filter paper on a seedling tray and place it in a 30 °C constant temperature incubator, with 100 seeds for each treatment. Track and observe the germination of maize seeds, record the number of germinated seeds, and analyze the promoting effect of the test strains on maize seed germination.

[0139] Germination potential % = (Number of germinated seeds at the peak germination stage / Number of tested seeds) × 100%;

[0140] Germination rate % = (Number of germinated seeds at the specified number of days / Number of tested seeds) × 100%.

[0141] After culturing for 7 days, the germination potential and germination rate of the control group seeds were 51.78% and 57.41% respectively, and the germination potential and germination rate of the test group seeds were 55.54% and 61.90% respectively. After treatment with Flavobacterium canadense ( Flavobacterium erigeronis ), the germination potential and germination rate increased by 3.76% and 4.49% respectively, indicating that Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 can alleviate saline-alkali inhibition and promote maize seed germination.

[0142] 2. Alleviate the growth stress of maize seedlings under saline-alkali conditions

[0143] Pick a single colony of Flavobacterium canadense ( Flavobacterium erigeronis ) 3-218 and inoculate it into a 500 mL conical flask containing 200 mL of R 2 A liquid medium, culture at 25 °C for 48 h. Centrifuge at 8000 rpm for 10 min, collect the bacterial cells, and resuspend them with R 2 A liquid medium to form a bacterial suspension with an OD 600 = 1.0 (using the uninoculated R 2A liquid medium was used as the control group for seed soaking. Maize (Zhengdan 958) seedlings with similar growth vigor were selected and transplanted into flower pots filled with 100 g of vermiculite substrate, with 3 plants per pot. A total of 9 pots were set up for both the experimental group and the control group (CK). Three days after transplanting and acclimatizing, saline-alkali stress treatment was carried out. Saline-alkali solution (NaCl, Na 2 SO 4 and NaHCO 3 , a mixed solution with a molar ratio of 1:1:1, and the final concentration of Na + was 168 mmol / L) of 50 mL was irrigated into each pot; on the second day after the stress treatment, root irrigation with the bacterial suspension was carried out. The roots of the maize seedlings in the treatment group were irrigated with the bacterial solution, 5 mL per plant, and the control group was irrigated with pure R 2 A liquid medium without inoculation. During the plant growth period, water was applied every 2 - 3 days, and the inoculant was supplemented on the 15th day. The experiment was carried out in a light incubator. The day and night temperatures were 25°C / 20°C respectively, and the sunshine duration was 14 h.

[0144] Figure 5 shows the growth status of maize seedlings under saline-alkali stress for 30 days. The final experimental data were the average values of each pot of plants.

[0145] Under saline-alkali stress, symptoms such as withering, curling, and shedding appeared on the leaves of maize seedlings. The plant height of the plants decreased significantly, and the overall showed a withering trend. Different degrees of salt damage occurred in different treatments. The results of the agronomic traits of maize seedlings Figure 6 are shown as follows. The fresh weight and dry weight of the maize seedlings in the experimental group (3 - 218) were 2.99 ± 0.22 g and 0.27 ± 0.07 g respectively, while the fresh weight and dry weight of the control group (CK) were 2.40 ± 0.19 g and 0.21 ± 0.06 g respectively. The fresh weight and dry weight of the experimental group were significantly higher than those of the control group (CK) ( P < 0.05). The plant height of the experimental group (3 - 218) was 23.31 ± 3.49 cm, which was significantly higher than that of the control (CK) ( P < 0.05), indicating that Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3 - 218 promoted the growth of maize plants under saline-alkali conditions.

[0146] Figure 7 are shown as follows. The stem diameter and chlorophyll content of the maize seedlings in the experimental group (3 - 218) were 3.48 ± 0.53 cm and 28.72 ± 2.54 SPAD respectively, while the stem diameter and chlorophyll content of the control group (CK) were 3.00 ± 0.58 cm and 27.16 ± 4.28 SPAD respectively. The stem diameter and chlorophyll content of the experimental group were better than those of the control group, and the stem diameter was significantly higher than that of the control group ( P < 0.05), indicating that Flavobacterium conyzicola ( Flavobacterium erigeronis3-218 can promote the growth of maize seedlings and the accumulation of chlorophyll under saline-alkali conditions, and significantly increase the stem diameter of maize seedlings.

[0147] To further study the ability of the strain to alleviate saline-alkali stress in maize seedlings, the activities of leaf catalase (CAT), peroxidase (POD), malondialdehyde (MDA) content, and proline (PRO) content were measured. An appropriate amount of maize seedling leaves were cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. According to the methods provided in the instructions of the catalase (CAT) activity detection kit (Solarbio, China), peroxidase (POD) activity detection kit (Solarbio, China), malondialdehyde (MDA) content detection kit (Solarbio, China), and proline (Pro) content detection kit (Solarbio, China), the CAT, POD, MDA, and PRO in the maize seedling leaves of the experimental group and the control group were extracted and measured.

[0148] The results are shown in Table 3. The MDA content in the experimental group (3-218) was higher than that in the control group (CK), but the difference was not significant ( P > 0.05), indicating that both groups were under a certain degree of stress. The higher content in the experimental group was presumably related to the plant stress response triggered when the strain, as an "invasive species", established an interaction with the crop; the CAT activity and POD activity in the experimental group (3-218) were significantly higher than those in the control group (CK) ( P < 0.05). The better CAT activity indicates that the plant has stronger antioxidant ability and resistance to stress, and the high activity of POD is also a sign of the plant's response to stress conditions. The above results show that Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3-218 can stimulate the plant to produce high-activity CAT and POD, enabling the plant to better cope with salt stress conditions and facilitating plant growth; at the same time, the PRO content in the experimental group (3-218) was significantly higher than that in the control group (CK) ( P < 0.05). As an important osmotic adjustment substance under stress conditions, the increase in its content indicates enhanced plant stress resistance. In summary, it is speculated that Flavobacterium conyzicola ( Flavobacterium erigeronis ) 3-218 alleviates the growth stress of maize seedlings under saline-alkali conditions and improves the saline-alkali adaptability of maize by inducing the plant to produce a large amount of PRO and high-activity CAT and POD.

[0149] Table 3. Enzyme activities and compound contents related to stress in maize seedling leaves at 30 d

[0150]

[0151] Note: Different lowercase letters in the same column indicate significant differences (P <0.05).

[0152] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Flavobacterium spp. Flavobacterium erigeronis ), its strain number is 3-218, and its registration number at the General Microbiology Center of China Culture Collection Administration is CGMCC No.31149.

2. The Flavobacterium spp. described in claim 1 ( Flavobacterium erigeronis ) is a culture of the bacterium of claim 1 ( Flavobacterium erigeronis ) Substances obtained by growing in bacterial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains the Flavobacterium sphaeroides described in claim 1 ( Flavobacterium erigeronis ) and / or the culture according to claim 2.

4. The bacterial agent according to claim 3, characterized in that: The bacterial agent is a bacterial agent for alleviating saline-alkali stress of plants.

5. The Flavobacterium spp. described in claim 1 ( Flavobacterium erigeronis ) or the culture according to claim 2 or any of the following uses of the bacterial agent according to claim 3 or 4: (A1) Secretion of siderophore; (A2) preparing a secretory siderophore product; (A3) Improve the activity of plant antioxidant enzymes under saline-alkali stress conditions; (A4) preparing products for increasing the activity of plant antioxidant enzymes under saline-alkali stress conditions; (A5) Increase the proline content of plants under saline-alkali stress conditions; (A6) preparing a product for increasing the proline content of plants under saline-alkali stress conditions; The plant is maize; The antioxidant enzyme is catalase or peroxidase.

6. The Flavobacterium spp. described in claim 1 ( Flavobacterium erigeronis ) or the culture according to claim 2 or any of the following uses of the bacterial agent according to claim 3 or 4: (B1) Promote plant growth under saline-alkali stress conditions; (B2) Preparation of products for promoting plant growth under saline-alkali stress conditions; (B3) Promote plant seed germination under saline-alkali stress conditions; (B4) preparing products for promoting plant seed germination under saline-alkali stress conditions; The plant is maize.

7. The Flavobacterium spp. of claim 1 ( Flavobacterium erigeronis ) or the culture according to claim 2 or any of the following uses of the bacterial agent according to claim 3 or 4: (C1) Improving plant germination potential and / or germination rate under saline-alkali stress conditions; (C2) preparing products for improving the germination potential and / or germination rate of plants under saline-alkali stress conditions; (C3) increase plant stem thickness under saline-alkali stress conditions; (C4) preparing a product for increasing the stem thickness of plants under saline-alkali stress conditions; (C5) increase plant fresh weight and / or dry weight under saline-alkali stress conditions; (C6) preparing a product for increasing the fresh weight and / or dry weight of plants under saline-alkali stress conditions; (C7) Increase plant height under saline-alkali stress conditions; (C8) preparing a product for increasing plant height under saline-alkali stress conditions; The plant is maize.

8. The Flavobacterium spp. described in claim 1 ( Flavobacterium erigeronis ) or the culture according to claim 2 or any of the following uses of the bacterial agent according to claim 3 or 4: (D1) Alleviate plant salinity and alkali stress; (D2) preparing products for alleviating saline-alkali stress in plants; The plant is maize.

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